Medication against breast cancer and related diseases

ABSTRACT

The present invention relates to novel uses of a steroid compound, which specifically binds to the androgen receptor (AR) but which is not metabolized by aromatase to a process for their preparation, to pharmaceutical compositions containing them, and to the use of said compounds for the prophylaxis and/or treatment of hormone-dependent or hormone receptor regulated cancers in mammals.

The present invention relates to novel uses of4,17β-dihydroxyandrost-4-ene-3-one (hereinafter 4-hydroxytestosterone)and its salts and esters, to a process for their preparation, topharmaceutical compositions containing them, and to the use of saidcompounds for the prophylaxis and treatment of certain diseases inmammals, in particular cancers.

After colorectal cancer breast cancer is the most frequent cancerdisease in the western world despite the fact that it affects almostexclusively women. In Germany, breast cancer amounts to about 20% of allcancer types diagnosed in women. The therapy in most of the casesconsists of surgical removal of the tumour (lumpectomy) or completeremoval of the affected breast if the size of the tumor prevents breastconserving therapy. In both cases the operation is followed by drugtherapy. A particular complication of breast carcinomas is the highpotential of metastatic spread into other organs particularly liver,brain, bones and the skin. Therefore the operation is followed by drugtherapy (adjuvant therapy). This therapy is also aimed at preventinglocal recurrence of the disease.

If the removed tumors were exhibiting estrogen receptors, antiestrogensare used (Tamoxifen or aromatase-inhibitors. The lack of estrogenreceptors leads to a postoperative drug therapy using cytostatic drugs.

Tamoxifen binds to the estrogen receptor (ER) and blocks the growthpromoting effect of estrogens. Aromatase-inhibitors block the final stepin the production of estrogens from androgenic precursors(c-19-steroids). They either bind irreversibly to the active site of theenzyme (aromatase-inactivators, i.e. steroidal aromatase-inhibitors:4-hydroxyandrostenedione (Formestane=Lentaron®), or Exemestane(Aromasin®)) or inhibit competitively the cytochrome p 450 part of theenzyme (nonsteroidal aromatase-inhibitors: Anastrozol (Arimidex®) andLetrozol (Femara®).

If the breast cancer exhibited an overexpression of the Her2neu gene,antibodies against this cell-surface protein are given intravenouslysuch as e.g. Trastuzumab (Herceptin®).

Basic and clinical data indicate that aromatized metabolites ofandrogens, i.e. the estrogens, are the hormones involved in thepathogenic cellular changes associated with the growth of somehormone-dependent cancers, such as breast, endometrial and ovariancarcinomas. Aromatase has recently been recognized as an enzyme that iscapable to synthesize estradiol directly in tumours.

Endogenous estrogens are ultimately formed from either androstenedioneor testosterone as immediate precursors. The local production ofestrogens in the breast tissue, especially in the tumour cells itself,had been regarded to be of great importance for the therapy of thedisease. In this context, aromatization of the steroidic ring Aperformed by the enzyme aromatase, and its inhibition had beenconsidered to play a major role in anti breast cancer approaches.

Prior to the present invention, conventional therapeutic approaches toaddress breast cancer in women—besides other concepts based on estrogenreceptor antagonists or therapeutic antibodies—therefore focused on anaromatase-inhibiting action itself. Accordingly, steroidal substanceswhich had been reported to be endowed with an aromatase-inhibitingaction have been described, for example Δ¹-testololactone [U.S. Pat. No.2,744,120], 4-hydroxy-androst-4-ene-3,17-dione and esters thereof [see,for example, U.S. Pat. No. 4,235,893],10-(1,2-propadienyl)-estr-4-ene-3,17-dione (U.S. Pat. No. 4,289,762],10-(2-propynyl)-estr-4-ene-3,17-dione [J. Amer. Chem. Soc., 103, 3221(1981) and U.S. Pat. No. 4,322,416], 19-thioandrostene derivatives(Europ. Pat. Appl. 100566), androsta-4,6-diene-3,17-dione,androsta-1,4,6-triene-3,17-dione [G.B. Pat Appl. 2,100,601A] andandrosta-1,4-diene-3,17-dione [Cancer Res. (Suppl.) 42, 3327 (1982)].

Clinical studies conducted with estrogen-receptor (ER) antagonists andaromatase inhibitors however showed that these compounds were not ableto exert a significant and sufficient inhibitory effect on tumourproliferation and/or growth. For example, Tamoxifen® based therapysuffers from drawbacks such as tachyphylaxy (therapy failure) and thefact that its effects in some cells are estrogen-like (e.g. risk forthrombosis and endometrial cancer) being the reason that Tamoxifen® cannot be administered for a period longer than 3 to 5 years. Newer drugssuch as aromatase inhibitors can in principle only be used inpost-menopausal women and, furthermore, are only effective onER-positive tumours. Estrogen receptor negative tumors have a worseprognosis and can be postoperatively only treated by cytostatic drugsand in some cases by intravenously given antibodies to the Her2neuantigen. “Hormonal” treatment with selective estrogen receptormodulators (SERMs) such as tamoxifen or raloxifen oraromatase-inhibitors has proven to be ineffective. “Hormonal” treatmentis lifelong and usually well tolerated. There is no “hormonal” treatmentfor ER-negative breast cancers so far.

It could be shown (J. Szelei et al., “Androgen-Induced Inhibition ofProliferation in Human Breast Cancer MCF7 Cells Transfected withAndrogen Receptor”, Endocrinol., 138, no. 4, 1406-1412, 1997) thatstable transfected cells expressing AR acquire the ability to respond toandrogen by evoking an end of cellular proliferation. A more recentreport (J. Ortmann et al., “Testosterone and 5α-dihydrotestosteroneinhibit in vitro growth of human breast cancer cell lines”, Gynecol.Endocrinol. 2002; 16:113-120) shows that testosterone and5α-dihydrotestosterone (DHT) can inhibit cell proliferation in certaincell lines. Testosterone itself has a similar effect. It clinicalusefulness however is hampered by the fact that it undergoes rapidmetabolization to either estradiol or dihydrotestosterone (DHT). Thelocal formation of estradiol from testosterone within the breast cancerby local aromatase activity promotes cancer-growth.

On the other hand, AR itself had been regarded as a modulator ofprocesses involved in differentiation, homeostasis, and development ofmale secondary characters. Thus, AR-directed treatment schemes had beenfocused on androgen-related diseases in men, including hypogonadism,male infertility, prostate cancer, delay of puberty, hirsutism,androgen-deficient diseases, androgen replacement in aging men, and malepattern baldness. In this context, a disclosure of U.S. Pat. No.2,762,818A does not go beyond using 4-hydroxytestosterone and itsesters, based on their androgenic and anabolic properties, to treat anandrogen deficiency status itself. There was however neither a purposenor a finding which would suggest an activity on breast cancer cells oron effects from which an efficacy against the disease conditionsaccording to the present invention could have been deduced. Further,US2003/0229063A addresses low androgen to estrogen ratios in men(leading to endocrine disorders) and only for this purpose attempts tomake use of 4-hydroxytestosterone based on an assertedaromatase-inhibiting effect alone.

In WO 2005/062760 incorporated herein in its entirety by way ofreference, possible roles of androgen receptor (AR) in prostatecarcinogenesis and breast cancer is discussed, and methods for breastcancer diagnosis by assaying the presence of AR are presented. However,in terms of therapeutic concepts, WO 2005/062760 is limited to controlAR itself, not androgen mediated activity, in the context of mammarygland development by inhibiting AR activity.

For certain indications, the present invention now provides successfulother therapeutic approaches involving androgen-receptor (AR) specificcompounds that exert testosterone-like effects (in particular anaboliceffects) in AR-positive cells and tissues and are not metabolized byaromatase.

According to the present invention, it was found that if a correlationbetween steroid compounds having specific affinity to the androgenreceptor (AR) and a certain receptor status of target cells is met, asurprising efficacy for specifically devised therapeutic treatments canbe achieved. Useful prophylaxis for corresponding disease conditions islikewise provided.

Thus, in an embodiment the present invention involves the use of asteroid compound, which specifically binds to the androgen receptor (AR)and preferably has a high affinity to AR but which is not metabolized byaromatase or 5-alpha reductase, for treating a patient in a therapy, orfor a prophylaxis in a condition selected from the group consisting ofbreast cancer, endometrial carcinoma, cervix carcinoma, epithelialovarian ovarian carcinoma, uterine (fallopian) carcinoma, benignproliferative change or alteration of the breast, fibroadenoma,macromastia (peri-postmenopausal), mastopathy, myoma, and secondary ormetastatic tumors derived therefrom, and shrinking breast tissue;wherein the patient has AR positive target cells or target tissue.

In the aforementioned embodiment, the patient may have estrogen receptor(ER) positive or estrogen receptor (ER) negative target cells or targettissue.

According to another embodiment of the present invention, the samecompound specifically binding to and preferably having high affinity tothe androgen receptor (AR) is used in the therapy or prophylaxis of thesame disease conditions, wherein however the patient has ER negativetarget cells or target tissue.

The concept of the present invention can be combined to provide evenbetter therapeutic prospects, i.e. in cases where the patient has ARpositive and ER negative target cells or target tissue.

If not known from other information or data, receptor status of targetcells or target tissues with respect to AR and/or ER can be determinedand, if desired, quantified by standard methods known to the personskilled in the art, including immunoassays involving AR- and/orER-specific antibodies, DNA and/or RNA hybridization assays or PCRamplification tests involving AR- and/or ER-specific nucleic acidprobes.

The compounds according to the invention shall be used in amountstherapeutically and/or prophylactically effective against the indicateddisease conditions. “Use” according to the present invention may includemethod of treatment or prophylaxis of the specified compounds, or of apharmaceutical composition containing the same as an active principletogether with a suitable carrier and/or diluent, for the describedindications, and it includes use in the preparation of saidpharmaceutical composition.

Based on the disclosed concept of the invention, there are also providedpreferred treatment or prophylaxis schemes involving the use of thecompounds of the invention per se, i.e. even independently from knowingthe receptor stats in advance. This is because some prior art treatmentschemes of breast cancer or related diseases typically presupposed thepresence of ER positive target cells. While this may be assumed for amajority of breast cancer cells, there is generally a risk of ERnegative cells being present which are not susceptive for effectivetreatment. Further there is a general risk of formation of metastasisderived from the primary cancers especially in cases where ER negativecancer cells were not successfully treated during treatment phase. Sincecompounds of the present invention have been demonstrated to be activeagainst ER negative cancer cells and tissues, new treatment modalitiesare opened, and they can be used with higher safety. Thus, in certainembodiments of the present invention the compounds disclosed herein areprovided for medical use, independent of the receptor status, inparticular:

(i) Application in a topical administration form, especially at the siteof the body affected by the disease to be treated or to be of interestprophylactically. This mode of administration allows to benefit from thedirect action via AR in the target cells and tissues, contrary to aneffect via a decrease of gonadotropin secretion (gonadotropins may evenpromote cancer growth by enhancing local estrogen production also in thetumour).(ii) Treatment or prophylaxis of postmenopausal women (which areparticularly prone to hormone-dependent disorders, and therefore forprophylaxis, or treating recurrence of breast cancer; or ofosteoporosis). Here again, the compounds of the present invention canexert the desired effects locally.(iii) Use for therapeutic or prophylactic indications of, alone or incombination:

-   -   ductal, medullary and lobular breast carcinoma (in particular        contralateral breast cancer, and/or in combination with the        aforementioned topical administration);    -   adjuvant and in particular neoadjuvant breast cancer treatment;    -   shrinking of breast cancer tissue;    -   prophylaxis against recurrence or remission of breast cancer;    -   skin metastases derived from breast cancer (in particular in        combination with the aforementioned topical administration).

Treatment or prophylaxis of these specific indications benefit from theparticular mode of activity of the compounds disclosed herein.

Particulars of the compounds to be used according to the invention willbe described in more detail.

The steroid compounds are required not to be metabolized by aromatase or5-alpha-reductase. Possible compounds satisfying these requirements arenon-aromatizable steroidal compounds and non-steroidal compoundsspecifically binding to AR, respectively. Although not being bound toany theory, various circumstances detrimental to the concept of thepresent invention could be attributed, if the compound was aromatizableby aromatase. First, cellular mechanisms invoked by the presence of sucha substrate could lead to an upregulation for enhanced expression of thearomatase enzyme and thus to an increased aromatase level at thecritical target tissue site. Second, aromatization of the compound boundto steroid-receptors in the cancer could lead to a compound exhibitingestrogen-like growth promoting activities. Third the resulting compoundcould diminish desired activities via the AR.

Further, the compounds are required to specifically bind to AR. Thecompound used according to the present invention preferably has a highbinding affinity to the androgen receptor (AR), i.e. having affinityspecific to AR in a range of IC₅₀≦50 nM, preferably IC₅₀≦100 nM and morepreferably IC₅₀≦50 nM, wherein IC₅₀ is defined as the concentration ofthe compound required to reduce specific binding of a referencecompound, 5α-dihydrotestosterone (DHT), by 50%. The IC₅₀— values can bedetermined by known methods using radioactively labelled DHT asreference compound, for example by a standard dextran-coated charcoaladsorption method as described by Raynaud et al., J. Steroid Biochem. 6,615-622 (1975), using 1 nM reference concentrations of radiolabeled[³H]-DHT, or by similar IC₅₀ determination methods described in theliterature. Because the concentration of AR in the target cell is verylow, typically in the nanomolar range approximately, differences inbinding constants to the order contemplated in the present invention aresignificant.

In consideration of both requirements, the compounds thereforepreferably share a common C-19 steroid structure, more preferably anandrost-4-en-17-ol-3-one structure, wherein the structure of thecompound may be further defined by a 1-en double bond and/or asubstituent bonded to the 17-ol (hydroxyl) group.

In a particular embodiment of the present invention, the compound isselected from compounds having the following formula:

wherein

-   a, b and c respectively denote, independently from each other, a    single bond or a double bond;-   R₁ is hydrogen or C₁ to C₆ alkyl;-   R₂ is hydrogen or OR₅, wherein R₅ is hydrogen or C₁ to C₆ straight    chain or branched alkyl;-   R₃ is, in case of c being a single bond, hydrogen or C₁ to C₆ alkyl,    or in case of c being a double bond, CHR₅, wherein R₅ is the same as    defined before;-   R₄ is hydrogen, C₁ to C₆ alkyl, phenyl unsubstituted or substituted    by C₁ to C₆ alkyl, COR₆ acyl group (R₆ being hydrogen; C₁ to C₆    straight chain or branched alkyl; phenyl or benzoyl respectively    unsubstituted or substituted by C₁ to C₆ alkyl), or any group    leading to hydroxyl upon biological metabolization or chemical    deprotection;    and pharmaceutically acceptable salts thereof.

In terms of a preferable consistency of being surely not aromatized, yetensuring strong affinity to AR, compounds are preferably selectedwherein b is a double bond, R₂ is hydrogen or hydroxyl and/or R₃ ismethylene group, and R₄ is hydrogen, C₁-C₆ alkyl or C₁-C₆ acyl group.Particularly preferred are 4,17β-dihydroxyandrost-4-en-3-one(4-hydroxytestosterone) (wherein a is single bond, b is double bond, R₂is OH and R₄ is hydrogen) and 17β-hydroxy-6-methylenandrost-1,4-diene(wherein a and b are double bonds, R₂ and R₄ respectively are hydrogen)and the corresponding esters (such as R₄ denoting COR₆ acyl group asdefined) and salts.

Compounds of the present invention also include those which aremetabolized to the above-defined compounds. For example,androst-3,17-dione structures (having 4-en- or 1,4-dien-bonds andoptionally 6-methylene group) are included as progenitor compounds whichupon metabolization in the body are metabolized to correspondingandrost-17-ol-3-one structures.

Moreover, it preferred that compounds are used which have predominantlyanabolic activities on target cells, rather than mere androgeniceffects. Further preferable are compounds which also display apoptoticeffects on target cells.

It has been devised that such properties can further contribute to aprophylactic and/or therapeutic efficacy against breast cancer andrelated disorders.

Although EP0307135A may partly disclose some compounds which may fallunder the aforementioned formula, its therapeutic concept deal witharomatase inhibition alone or, if related to a possible androgenicactivity, with an inhibitory effect on estrogen biosynthesis through adecrease in gonadotropin secretion (i.e. systemic effects, necessitatingactions via generative glands, ovaries, LH-associated effects and thelike). The concepts of the present invention however differ, and thuscompounds are selected by distinctly different selection criteria interms of effects exerted directly at the site of interest byconsiderations directed to, alone or in combination, receptor status oftarget cells or tissues; mode of administration; patient group; andcertain indicated disease or prophylactic conditions.

The compounds of the present invention exhibit androgen-receptor (AR)binding affinity. In most cells, except breast cancer cells they arecapable of inducing an anabolic effect, provided they exhibit AR. Inbreast cancer cells they induce apoptosis. These compounds are neithersubstrates for nor metabolic products from aromatase and thus do notshare the negative effects of substances being metabolized by aromatase.Furthermore, the compounds of the present invention are not substratesfor 5α-reductase. As these substances cannot be that easily metabolisedas, for example, testosterone, their inhibitory effect on the tumourcell is greatly facilitated and they additionally exert an apoptoticeffect on the tumour cell itself. Therefore, these substances arepresumably more efficient in the treatment of breast cancer cells thanthe line of currently used substances to treat both types of breastcancer. Another beneficial effect of this prophylactic or therapeuticconcept is that even in estrogen receptor negative (ER-negative) tumoursthere is a high probability of the cells to be AR-positive and thereforeeven these tumours currently treated only with cytostatic drugs can bepharmacologically addressed by 4-hydroxytestosterone, thereby increasingthe range of hormonal breast cancer therapy significantly.

Since the compounds of the present invention are AR specific compounds,but are not metabolized by aromatase, they can fully exert their actionsand effects associated with the specifically defined therapeutic orprophylactic uses via AR. This makes the concept of the presentlyselected compounds distinct over the prior art.

In experiments carried out with the compounds of the present inventionit could be shown that these substances had excellent skin penetrationcapabilities so that a sufficient tumour inhibition could be achieved bysimple topic administration of e.g. an ointment, lotion or cream etc.comprising a therapeutically or prophylactically effective amount of acompound according to the present invention to an area of a patient inneed of treatment. After topical administration, the compound(s)penetrate through the skin and concentrate in the fatty tissue.

In the case of breast cancer, the medicament can be applied locally tothe skin, such that the preferably strongly lipophilic active compoundis transdermally absorbed and thus brought locally to the intended siteof action. The active compound concentrates in the periductal fattytissue. In a long-term treatment, the fatty matter of the treated breastis markedly reduced. This reduction decreases the quantity ofestrogen-forming cells having estrogen-forming competence. Thelipophilicity and hydrophobicity of the active compound has the resultthat the active compound is exclusively concentrated locally in thefatty tissue and thus cannot display any significant systemic action.

According to a preferred embodiment of the present invention it isintended to apply the medicament topically immediately on or in thevicinity of the intended site of action. According to the invention, theresult achieved is an adequate local active compound level in the tissueat risk (in prophylaxis) or in the diseased tissue (in therapy) withouta noticeable absorption of the active compound taking place in the bloodcirculation. The benefit of this embodiment thus not only relies in thetopical application per se, but in the local topical application in sucha way that the active compound concentrates immediately in the tissue atrisk and/or diseased tissue, and not indirectly via the bloodcirculation.

Owing to the primary target disease to be treated as described above,especially for breast cancer etc., a local topic treatment orprophylactic application is predominantly carried out with femalepatients and particularly on breast skin of female patients, althoughthe same therapeutic principle applies also to male breast cancer.

If metastasizing carcinomas are also to be treated and/or prophylaxis isto be carried out against these, the medicament according to theinvention can be applied topically to the intended site of action insuch an amount that a noticeable absorption in the blood circulationadditionally takes place and a serum level thus builds up which alsotransports the active compound to metastases. In this use too, a localabsorption on or in the vicinity of the intended site of action, notablywhere metastasizing carcinomas exist, also primarily takes place.

An important advantage of the invention is the possibility of also usingthe medicament for breast cancer prophylaxis. A particularlyadvantageous possibility of employment is so-called secondaryprophylaxis. In female or male patients in whom a breast cancer has beendiagnosed and treated, there is a certain risk of recurrence at the samesite and also a high risk of a further carcinoma in the contralateralbreast. In both cases the endowment of the second cancer with ER is notpredictable. The operated and the contralateral breast can then betreated prophylactically with the medicament according to the invention.

It will prevent also the appearance of a ER negative cancer(particularly common in patients suffering from a familiarpredisposition for breast cancer).

In the case of so-called high-risk women, primary prophylaxis can beperformed. The selection criteria which can be used for such a high-riskgroup are, for example, the facts that at least one female or malerelative of first degree on the mother's side is or has been sufferingfrom breast cancer on one side before the 45^(th) year of life orbilaterally, or that on the mother's side at least one female or malerelative of first degree and an additional female or male relative areor have been suffering from breast cancer.

In the majority of theses cases the underlying cause of the disease is agenetic defect in one of the two known “breast cancer genes” BRCA1 andBRCA2.

Since the local application according to a preferred embodiment of theinvention virtually completely avoids possible systemic side effects ofthe active compound on account of its hydrophobicity and high safetyprofile, the indication for primary prophylaxis can be made relativelygenerously already in the presence of tissue having a comparatively lowor average risk. Prophylaxis can be started long time before signs andsymptoms of breast cancer may be occurring.

In the preferred topical administration the compound or compositionaccording to the invention is preferably applied over a relatively longperiod of time (if needed up to lifelong) and application is carriedout, for example, once or twice per day. Since the active substances ofthe invention intended to be applied topically (locally) are preferablyprovided in lipid-soluble form, they can easily penetrate into the fattytissue and locally exert their prophylactic and/or therapeutic effect.

Since the active compounds administered topically according to theinvention remain localized in the fatty tissue of the breast and displaytheir intended action there on account of their lipid solubility, anypossible side effects induced by systemic application are eliminated.This reduction or exclusion of side effects allows significantly widerprophylactic use. The medicament according to the invention can beapplied by patients themselves and frequent visits to the doctor forthis purpose are not necessary.

The advantages described above further contribute to the particularsuitability of the compounds of the present invention for treatinggenerally breast carcinoma, and more beneficially treating the same bytopical administration; contralateral breast cancer; adjuvant and inparticular neoadjuvant breast cancer treatment; shrinking of breastcancer tissue; prophylaxis against recurrence or remission of breastcancer; skin metastases derived from breast cancer; and the likeconditions.

In particular for topic administration, a medicament formulatedaccording to the invention preferably also contains formestane.

Formestane derivatives such as, for example, acetylated formestane (forexample 4-O-acetylandrost-4-ene-3,17-dione) are likewise preferablyutilizable. The acetylation of the formestane increases itshydrophilicity and thus skin penetration significantly. Since the acetylgroup is hydrolysed under the conditions prevailing in the subcutaneousregion after passage through the skin, the actual active compoundformestane is formed again in situ. When using such an acetylatedformestane, a precursor of the actual active compound penetrating betterthrough the skin is thus applied and it has been recognized that theactual active compound is formed in situ subcutaneously from thisprecursor.

As a rule for topic administration, the active compounds used accordingto the invention are lipid-soluble and highly suitable for topicalapplication. As already described above, the concentration in the fattytissue of the breast avoids systemic side effects. To improve the skinpenetration, substances known in the prior art which promote this can beadded to the medicament according to the invention, for exampleHyaluronidases, Dimethylsorbitol or DMSO (dimethyl sulphoxide).

In particular, the topic administration is mainly indicated for primaryprevention (prophylaxis), secondary prevention (prophylaxis) afterbreast conserving therapy, accompanied by any form of adjuvant drugtherapy, after operation for ‘ductal carcinoma in situ’ (DCIS, which isusually not followed by any adjuvant therapy) and prophylaxis ofcontralateral breast cancer.

Primary prevention relates to the prophylactic treatment of healthyindividuals bearing a certain risk to develop cancer. Since thecompounds and pharmaceutical compositions of the invention are not asubstrate of aromatase and thus cannot be metabolized to estrogens, andare not virilizing, they can also be used in the primary prevention ofbreast cancer in premenopausal women. In secondary prevention, thecompounds and compositions may be used alone or in combination with e.g.Tamoxifen, aromatase inhibitors, herceptine and/or cytostatic drugs,wherein any of such combinations would enlarge the spectrum of treatablecell types.

Thus, the present invention beneficially provides also a combinationmedication comprising, in common or separate administration forms forsimultaneous or subsequent medical use, a compound of the presentinvention and a substance selected from the group consisting ofestrogen-receptor antagonists, aromatase inhibitors different from thecompounds of the invention disclosed herein, cytostatics and antibodiesagainst Her2neu.

The concept of “combination medication” according to the presentinvention includes that the compound according to the invention on theone hand and the other aforementioned substance on the other hand arecomprised in one common formulation or administration form, or inseparate formulations or administration forms. Correspondingly, thepharmaceutical medication or preparation according to the invention maybe embodied by one formulation in the form of a unit composition, oralternatively as “kit-of-part” products sharing the common purpose ofbeing used separately, simultaneously, or sequentially.

The present invention is based on the findings that4,17β-dihydroxyandrost-4-ene-3-one (hereinafter 4-hydroxy-testosterone),and compounds similar thereto in view of common structural andfunctional features as defined above, can successfully be used as apharmaceutically active substance. In particular, it could be shown thatpharmaceutical compositions containing a suitable carrier and/or diluentand, as an active principle, 4-hydroxytestosterone, can be used in theprophylaxis and/or treatment of breast cancer (in particular ductal,medullary and lobular breast carcinoma), endometrial carcinoma, cervixcarcinoma, epithelial ovarian carcinoma, uterine (fallopian) carcinoma,benign proliferative change or alteration of the breast, fibroadenoma,macromastia (peri-postmenopausal), mastopathy, myoma, and secondary ormetastatic tumors derived therefrom.

Hence, the present invention likewise provides the use of4,17β-dihydroxyandrost-4-ene-3-one (4-hydroxytestosterone) and similarcompounds as defined above in the preparation of a pharmaceuticalcomposition for the (clinical) prophylaxis and/or treatment of the abovehormone-dependent or hormone receptor regulated cancers in mammals.

In general, the compounds and pharmaceutical preparations of theinvention can be administered topically, orally, rectally, parenterally(e.g. intramuscularly or by intravenous injection or infusion), and/orin the form of a depot preparation.

A particularly preferred compound of the present invention,4-hydroxytestosterone, is disclosed in e.g. U.S. Pat. No. 2,762,818 Aand commercially available (e.g. from Bulk Nutrition, Graham, N.C.,USA—see bulknutrition.com for further information; WINKOS GmbH D-79189Bad Krozingen, Del.). The derivatives, in particular salts and esters ofthe preferred 4,17β-dihydroxyandrost-4-ene-3-one include suitable estergroups, such as straight chain, branched chain or cyclic or aromaticacyl groups like formyl, acetyl, propionyl, butyryl, pentanoyl,hexanoyl, heptanoyl and benzoyl, but are not limited thereto. The esterscan be formed with the 4 and/or 17 hydroxy group, preferably with the 17hydroxy group. Its salts and esters can also be prepared by knownmethods (see e.g. U.S. Pat. No. 2,762,818 A).

The compounds and pharmaceutical preparations of the invention areanabolic steroids and exert their testosterone-like effects upon bindingto androgen receptors displayed in cells and tissues being AR-positive.

In view of their high therapeutic index, the compounds of the inventioncan be used safely in medicine. For example, the approximate acutetoxicity (LD₅₀) of the compounds of the invention in the mouse,determined by single administration of increasing doses and measured onthe seventh day after the treatment was found to be negligible.

The compounds and pharmaceutical preparations or compositions of theinvention can be administered in a variety of dosage forms, e.g.topically, in the form of an ointment, a cream, a lotion, a gel, aspray, or a transdermal plaster, also comprising depot dosage forms(including pellets), orally, in the form of tablets, capsules, sugar orfilm coated tablets, liquid solutions or suspensions: rectally, in theform of suppositories; parenterally, e.g. intramuscularly, or byintravenous injection or infusion. According to a preferred embodiment,the compounds of the invention are designed for topical administration.

The dosage depends on the age, weight, conditions of the patient andadministration route; for example the dosage adopted for oraladministration to adult humans may range from about 10 to about 150-1000mg pro dose, from 1 to 5 times daily.

The invention includes pharmaceutical preparations or compositionscomprising a compound of the invention in association with apharmaceutically acceptable excipient (which can be a carrier ordiluent).

The pharmaceutical compositions containing the compounds of theinvention are usually prepared following conventional methods and areadministered in a pharmaceutically suitable form.

For topical administration, the medicament is preferably formulated asan ointment, cream, gel, emulsion or lotion. Formulation as a powder oroil is also conceivable. Formulation bases are familiar to the personskilled in the art from the cosmetic and pharmaceutical industry and donot need to be explained here in greater detail. For example, vegetableoils and fats such as almond oil, peanut oil, olive oil, peach kerneloil, castor oil, plant extracts, ethereal oils; furthermore vegetablewaxes and synthetic and animal oils, fats or waxes; lecithin, lanolinalcohols, carotene, fragrances, mono- or polyhydric alcohols, urea,preservatives and colourants etc. can be used. Formulation as anoil-in-water or water-in-oil emulsion is preferred.

The active compound content of a suitable medicament can be between0.0001 and 20% by weight, preferably 0.6% until 10% by weight, furtherpreferably 1 and 5% by weight, of the compound according to theinvention. A customary range is 0.6 to 5% by weight.

If substances are admixed to promote skin penetration, their content,when using hyaluronidases, can be, for example, between 0.01 anti 1% byweight, preferably 0.05 anti 0.2% by weight, when using dimethylsorbitolor DMSO between 1 and 25% by weight, preferably 5 and 10% by weight

For example the solid oral forms may contain, together with the activecompound, diluents, e.g. lactose, dextrose, saccharose, cellulose, cornstarch or potato starch; lubricants, e.g. silica, talc, stearic acid,magnesium or calcium stearate, and/or polyethylene glycols; bindingagents, e.g. starches, arabic gums, gelatine, methylcellulose,carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents,e.g. a starch, alginic acid, alginates or sodium starch glycolate;effervescing mixtures; dyestuffs, sweeteners; wetting agents, such aslecithin, polysorbates, laurylsulphates; and, in general, non-toxic andpharmacologically inactive substances used in pharmaceuticalformulations. Said pharmaceutical preparations may be manufactured inknown manner, for example, by means of mixing, granulating, tabletting,sugar-coating, or film-coating processes. The liquid dispersions fororal administration may be e.g. syrups, emulsions and suspensions.

The syrups may contain as carrier, for example, saccharose or saccharosewith glycerine and/or mannitol and/or sorbitol.

The suspensions and the emulsions may contain as carrier, for example, anatural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol.

The suspensions or solutions for intramuscular injections may contain,together with the active compound, a pharmaceutically acceptablecarrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g.propylene glycol, and if desired, a suitable amount of lidocainehydrochloride.

The solutions for intravenous injections or infusions may contain ascarrier, for example, sterile water or preferably they may be in theform of sterile, aqueous, isotonic saline solutions.

The suppositories may contain together with the active compound apharmaceutically acceptable carrier, e.g. cocoa-butter, polyethyleneglycol, a polyoxyethylene sorbitan fatty acid ester surfactant orlecithin.

In cases where the target tumour is estrogen-receptor (ER) positive, thecompounds of the invention can also be administered together with knownestrogen-receptor antagonists (such as Tamoxifen®) and/or knownaromatase inhibitors and/or known cytostatics. Thus, a pharmaceuticalcomposition of the invention may also comprise an effective amount of(a) known compound(s) exerting estrogen-receptor antagonistic and/oraromatase inhibitory activity (compounds different from the compounds ofthe invention disclosed herein) and/or cytostatic activity. According toa preferred embodiment, a combination of 4-hydroxytestosterone and asuitable cytostatic or combination of cytostatics is provided, inparticular, in form of a preparation that can be administered topically(e.g. cream). Examples of suitable aromatase inhibitors are e.g.disclosed in US 2004/0018991 A1 and include steroidal aromataseinhibitors such as formestane and lentaron, exemestane, MDL 18962, 7alpha-substituted androstenedione derivatives, ATD, 10-oxirane and10-thiirane-substituted androgens, atamestan, etc., and non-steroidalaromatase inhibitors such as vorazole, arimidex, letrozole, fadrozole,and rogletimide. With regard to the designations of these substances aswell as their availability, see for example “Red List”, Editio Cantor,Aulendorf (DE, 1999).

Examples of suitable cytostatics and cytostatic combinations includee.g. AC±T—Adriamycin (chemical name: doxorubicin) with cyclophosphamide(brand name: Cytoxan), with or without Taxol (chemical name: paclitaxel)or Taxotere (chemical name: docetaxol); CMF—cyclophosphamide,methotrexate, and fluoro-uracil (“5-FU” or 5-fluorouracil);CAF—cyclophosphamide, Adriamycin, and fluorouracil (“5-FU” or5-fluorouracil); CEF—cyclophosphamide, Epirubicin (similar toAdriamycin), and fluorouracil (“5-FU” or 5-fluorouracil);FAC—fluorouracil (“5-FU” or 5-fluorouracil), Adriamycin, andcyclophosphamide.

In general, and in particular if the target tissue is estrogen-receptornegative, the compounds of the invention can also be administeredtogether with antibodies against Her2neu such as e.g. Trastuzumab(Herceptin®). Thus, a pharmaceutical composition of the invention mayalso comprise an effective amount of known compounds exertingantagonistic Her2neu activity.

The following examples illustrate but do not limit the invention.

EXAMPLE 1

The compound of the present invention can be synthesized as follows.

In a first step, 2.5 g testosterone is dissolved in 100 ml cold MeOH.After adding 9 ml NaOH (2%) and 17 ml H₂O₂ (30%) the mixture is stirredfor 24 h at 4° C. The resulting epoxids are precipitated with ice-water.

In a second step, 2 g of the dry epoxids are dissolved in 200 ml aceticacid containing 2% H₂SO₄. The solution is stirred for 4 h at roomtemperature. The reaction products are precipitated with ice-water.

Thereafter, the reaction products are washed with 1% NaOH solution tohydrolyse the acetyl esters. The total yield of pure4-hydroxytestosterone is in the range of 40-50%.

EXAMPLE 2

A cream for topical administration according to the invention can beformulated in conventional manner using the following amounts ofingredients. The amounts are given per 100 g of cream:

4-hydroxy-17β-acetyl-androst-4-en-3-one 4.5 g Cetearyl alcohol 7.5 gParaffin wax 3.0 g Sodium carbomer 2.5 g Isopropyl myristate 6.0 gSorbitan monostearate 1.0 g Polysorbate 20 3.0 g Stearyl alcohol 2.0 gDMSO 5.0 g Purified water qs 100.0 g 

The resulting cream can be given topically on the skin above affectedbreast tissue of a female or male patient.

EXAMPLE 3

A gel according to the invention can be formulated in conventionalmanner using the following amounts of ingredients. The amounts are givenper 100 g of gel:

4-hydroxytestosterone 2.5 g Ethanol 95. degree 70.0 g  Carbopol 980 0.5g Isopropyl myristate 2.5 g Triethanolamine 0.5 g Purified water qs100.0 g 

EXAMPLE 4

A solution for use as a spray according to the invention is prepared inconventional manner using the following formulation. The amounts aregiven per 100 g of solution:

4-hydroxytestosterone 2.5 g Ethanol 95. degree. 70.0 g  Isopropylmyristate 2.5 g Purified water qs 100.0 g 

EXAMPLE 5

Tablets each weighing 0.150 g and containing 25 mg of the activesubstance, can be manufactured as follows:

Composition (for 10000 tablets) 4,17β-dihydroxyandrost-4-ene-3-one 250 gLactose 800 g Corn starch 415 g Talc Powder  30 g Magnesium stearate  5g

The 4,17β-dihydroxyandrost-4-ene-3-one, the lactose and half the cornstarch are mixed; the mixture is then forced through a sieve of 0.5 mmmesh size. Corn starch (10 g) is suspended in warm water (90 ml) and theresulting paste is used to granulate the powder. The granulate is dried,comminuted on a sieve of 1.4 mm mesh size, then the remaining quantityof starch, talc and magnesium stearate are added, carefully mixed andprocessed into tablets.

EXAMPLE 6

Capsules, each dosed at 0.200 g and containing 20 mg of the activesubstance can be prepared.

Composition for 500 capsules: 4,17β-dihydroxyandrost-4-ene-3-one 10 gLactose 80 g Corn starch  5 g Magnesium stearate  5 g

This formulation is encapsulated in two-piece hard gelatine capsules anddosed at 0.200 g for each capsule.

EXAMPLE 7

An ointment for topical administration according to the invention can beformulated in conventional manner using the following amounts ofingredients. The amounts are given per 100 g of ointment:

17β-hydroxy-6-methylenandrost-1,4-diene 2.5 g Propylene glycol 25.0 g Isopropyl myristate 6.0 g Sorbitan monostearate 1.0 g Polysorbate 80 2.0g Stearyl alcohol 2.0 g Hyaluronic acid 0.1 g Purified water qs 100.0 g 

The resulting ointment can be given topically on the skin above affectedbreast tissue of a female patient.

EXAMPLE 8

A gel for simultaneous combination topical administration according tothe invention can be formulated in conventional manner using thefollowing amounts of ingredients. The amounts are given per 100 g ofgel:

4-hydroxytestosterone 2.75 g Formestane 1.25 g Ethanol 80% 10.0 gCarbopol 934 P  8.0 g PEG 400  2.5 g urea  3.0 g ethyl oleate  0.5 gpurified water to  100 g

EXAMPLE 9

Tablets for simultaneous combination medication according to theinvention can be formulated according to Example 5, except that half ofthe amount of 4,17β-dihydroxyandrost-4-ene-3-one is replaced byFormestane.

EXAMPLE 10

MCF-7 cells were grown in RPMI 1640 medium at 37° C. to a cell number ofabout 1*10⁵ cells/ml. Then, in quadruple identical MCF-7 cell samples,there were added either dihydro-testosterone (DHT) or4-hydroxytestosterone (4-OHT), to obtain different concentrationsthereof, namely 10⁻⁷ M, 10⁻⁸ M or 10⁻⁹ M, and further growth of cellswere determined by counting of vital cells. During the course ofincubation at 37° C., cell counting were performed after 3, 6 and 9days. For control, growth of MCF-7 cells were determined withoutaddition of an active agent.

The results on an inhibition of MCF-7 cell proliferation are shown inFIGS. 1A, 1B and 1C for the compound concentrations of 10⁻⁷ M, 10⁻⁸ Mand 10⁻⁹ M of either DHT or 4-OHT, respectively, and compared with thecontrol. The results demonstrate a significantly enhanced inhibitoryeffect on MCF-7 cell proliferation in case of using the compound4-hydroxy-testosterone according to the present invention, significantlystronger than 4-dihydrotestosterone and particularly stronger than thecontrol.

The surprisingly enhanced efficacy of 4-OHT compared to DHT can beexplained by the effects, that 4-OHT can specifically bind strongly tothe androgen receptor (AR) and eliciting anabolic/apoptotic effectsrather than androgenic effects. DHT on the other hand preferentiallyevokes androgenic effects.

EXAMPLE 11

Double blind clinical tests were conducted with a group of 10 femalepatients. In this clinical testing, particular attention was paid tohormone receptor statuses of patients' target breast cancer tissueregarding progesterone receptor (PR) and in particular regardingestrogen receptor (ER); The cancers of the patients were assumed to bepredominantly androgen receptor (AR) positive. Among this group, sixpatients had histologically ascertained malign breast carcinoma (C.mammae). The tumors of 5 patients of this investigated sub-group werefound to be progesterone receptor (PR) negative and, in particular,estrogen receptor (ER) negative (status level 0 (zero)). With respect toHer-2-neu, three patients did show a negative status, one had a moderate(level 1) and another had a strong (level 2) receptor status. In a sixthpatient, the presence of a malign breast carcinoma was ascertained, butthe amount of cancer tissue was too low to determine receptor status.

The remaining four patients did not show a malign disease, but appearedas having normal or mastopathic breast tissue.

All 10 female patients were topically and locally treated using4-hydroxytestosterone as active agent, by administering of a cream asdescribed in Example 2 locally to target skin portions of the breasts.After continuing daily topical administration once a day during anadministration period of 3 months, clinical investigations ontherapeutic effects were performed.

The results of the 6 patients having malign breast carcinoma are shownin the following Table 1, together with the receptor status determinedfor patient nos. 1 to 5.

Patient Clinical No. Age ER PR Her2neu Remission # 1 62 yrs. 0 0 0 yes #2 54 yrs. 0 0 1 pending # 3 87 yrs. 0 0 0 yes # 4 68 yrs. 0 0 2 pending# 5 57 yrs. 0 0 0 pending # 6 50 yrs. Ø Ø Ø yes

Among those women so far subjected to post-treatment investigations,three patients did show a noticeable remission of breast cancer tissuesas determined by mammography.

Though receptor status of the tumor of the sixth patient of this cancersub-group is unknown, there was also a clear clinical remissionascertained in this case.

With respect to the remaining 4 patients having non-malign diseasestates, it is to be noted that there is a clear tendency for tissuereduction of the apparently normal or mastopathic appearing tissues inthe course of topical administration.

The results of this clinical study show two main important effects ofthe pharmaceutical composition according to the present inventioncomprising 4-hydroxytestosterone (4-OHT). Firstly, 4-OHT shows anexcellent efficacy in shrinking cancerous tissues. Secondly, and stillmore surprisingly, even ER negative cancer cells can be effectivelytreated.

Taken together, the findings made in the present invention support theconcept that 4-OHT can efficiently act via the AR, and that apoptosiscan eventually be realized.

The findings of the present invention thereby make valuable therapeuticand/or prophylactic treatments feasible. Among other useful applicationsincluding adjuvant therapy, neoadjuvant therapy is of particularimportance by making use of the substantial reduction of tumour volumeto assist or enable subsequent surgical treatment schemes.

EXAMPLE 12

A 42 years old patient with bilateral breast-cancer and skin metastasesafter surgery. was treated daily with a cream corresponding to example2. After 4 weeks of treatment the skin metastases have disappeared.

EXAMPLE 13

A 50 years old patient with large breast cancer (diameter: 12 cm)massive tumour-spread and metastatic liver disease was treated bypartial tumor-resection and subsequent daily application of a creamcorresponding to example 2. The patient exhibited under treatment nofurther tumor growth and even a reduction of nonresectable tumour-mass.

EXAMPLE 14

A 74 years old patient with advanced breast cancer and tumour-spread tothe lymphatics was treated daily for 10 weeks. A substantial reductionof tumor volume occurred and in the affected lymph nodes no tumor cellswere present anymore.

1-9. (canceled)
 10. A method of prophylaxis against or treatment ofbreast cancer, endometrial carcinoma, cervix carcinoma, epithelialovarian carcinoma, uterine carcinoma, benign proliferative change oralteration of the breast, fibroadenoma, macromastia, mastopathy, myoma,secondary or metastatic tumors derived therefrom, and shrinking breasttissue, comprising administering patients in need of such treatment andhaving AR positive target cells or target tissue, and/or ER negativetarget cells or target tissue an effective amount of steroid compound,which specifically binds to the androgen receptor (AR) but which is notmetabolized by aromatase.
 11. Method according to claim 10, wherein saidsteroid compound is administered together with (an) effective amount(s)of at least one substance selected from the group consisting ofestrogen-receptor antagonists, aromatase inhibitors other than saidsteroid compound, cytostatics and antibodies against Her2neu.
 12. Methodaccording to claim 10, wherein the administration is topical.
 14. Amethod of prophylaxis against or treatment in the prophylaxis ortreatment under conditions selected from: (i) formulation in a topicaladministration form; (ii) treatment or prophylaxis of postmenopausalwomen; (iii) treatment or prophylaxis of, alone or in combination:contralateral breast cancer, adjuvant or neoadjuvant breast cancertreatment; shrinking of breast cancer tissue; prophylaxis againstrecurrence of breast cancer; skin metastases derived from breast cancer,comprising administering patients in need of such prophylaxis ortreatment an effective amount of a compound having the followingformula:

wherein a, b and c respectively denote, independently from each other, asingle bond or a double bond; R₁ is hydrogen or C₁ to C₆ alkyl; R₂ isOR₅, wherein R₅ is hydrogen or C₁ to C₆ straight chain or branchedalkyl; R₃ is, in case of c being a single bond, hydrogen or C₁ to C₆alkyl, or in case of c being a double bond, CHR₅, wherein R₅ is the sameas defined before; R₄ is hydrogen, C₁ to C₆ alkyl, unsubstituted or C₁to C₆ alkyl-substituted phenyl, COR₆ acyl group (R₆ being hydrogen; C₁to C₆ straight chain or branched alkyl; phenyl or benzoyl respectivelyunsubstituted or substituted by C₁ to C₆ alkyl), or any group leading tohydroxyl upon biological metabolization or chemical deprotection; orpharmaceutically acceptable salts thereof; or a pharmaceuticalcomposition containing said compound as an active principle, a suitablecarrier and/or diluent.
 14. The method according to claim 13, or thepharmaceutical composition containing the same, wherein the compound is4,17β-dihydroxyandrost-4-ene-3-one.
 15. Combination medicationcomprising, in common or separate administration forms for simultaneousor subsequent medical use, (I) a compound having the following formula:

wherein a, b and c respectively denote, independently from each other, asingle bond or a double bond; R₁ is hydrogen or C₁ to C₆ alkyl; R₂ isOR₅, wherein R₅ is hydrogen or C₁ to C₆ straight chain or branchedalkyl; R₃ is, in case of c being a single bond, hydrogen or C₁ to C₆alkyl, or in case of c being a double bond, CHR₅, wherein R₅ is the sameas defined before; R₄ is hydrogen, C₁ to C₆ alkyl, phenyl unsubstitutedor substituted by C₁ to C₆ alkyl, COR₆ acyl group (R₆ being hydrogen; C₁to C₆ straight chain or branched alkyl; phenyl or benzoyl respectivelyunsubstituted or substituted by C₁ to C₆ alkyl), or any group leading tohydroxyl upon biological metabolization or chemical deprotection; orpharmaceutically acceptable salts thereof; and (II) a substance selectedfrom the group consisting of estrogen-receptor antagonists, aromataseinhibitors different from said compound defined under item (I),cytostatics and antibodies against Her2neu.
 16. Combination medicationaccording to claim 15, formulated for topic administration.